Malignant transformation of cells is virtually always accompanied by alterations in the posttranslational modifications (PTMs) of proteins, and one of the best documented examples is the abundant mucin-type O-glycosylation (hereafter referred to as O-glycosylation) found on mucins and other O-glycoproteins (Tarp and Clausen 2008). Tumor-associated changes in expression of O-glycoproteins and/or in their aberrant glycosylation, create a diverse set of unusual molecular structures found on the surface of cancer cells as well as in secretions. These molecular structures generally represent glycoproteins with truncated immature O-glycans, to which the immune system of man is not normally exposed except, in some cases, as biosynthetic intermediates and then only in the secretory pathway. Therefore, these structures may represent a different form of tumor-associated antigens (one not necessarily based on differential expression level or sequence mutation), to which individuals lack immunological tolerance and thus provoke both auto-antibodies and cell mediated immunity (Anderton 2004; Doyle and Mamula 2005; Doyle and Mamula 2001).
Several mouse antibodies have been isolated and characterized as having unique binding specificity for combined glycopeptide epitopes that include both the peptide sequence as well as the aberrant PTM (hereinafter “APTM”) for efficient binding. Examples include monoclonal antibodies that specifically recognize distinct O-glycopeptides from MUC2, MUC1, and other glycoproteins (Reis et al. 1998; Sorensen et al. 2006; Danielczyk et al. 2006; Dian et al. 2009; Li et al. 2009; Takeuchi et al. 2002; Clark et al. 1998).
There exist examples of human antibodies with selective or specific reactivity with PTM-modified proteins, but these are generally limited to inflammatory and autoimmune diseases (Anderton 2004; Doyle and Mamula 2005; Doyle and Mamula 2001). Furthermore, human hydridoma technologies have identified natural IgM antibodies that react with glycoforms of proteins but the nature of the epitopes has not been fully clarified (Rauschert et al. 2008; Vollmers and Brandlein 2009; Brandlein et al. 2004a; Brandlein et al. 2004b; Rasmussen and Ditzel 2009). A major drawback of current technologies to screen for auto-antibodies directed against APTM proteins is the lack of high through-put methods for identifying antigens, more particularly epitopes, for generating pertinent antigens and for screening such antigens.
In principle, cancer-associated auto-antibodies represent appealing potential biomarkers. Auto-antibodies may develop early in carcinogenesis, at the time tumor-associated antigens appear on premalignant or malignant lesions. Antibody responses can produce relatively high concentrations in circulation with a long circulation time, and they can be detected with sensitive and specific methods (see, (Lu et al. 2008; Anderson and Labaer 2005)). In contrast, antigens produced by small premalignant or malignant lesions are generally produced in vanishingly small levels that due to dilution and clearance from blood may not be detectable by conventional techniques. Discovery and characterization of specific auto-antibodies to cancer antigens have been undertaken using different approaches in the past. Classical studies identified such antibodies reactive with tumor cells, tissues, or isolated proteins (Kawabata et al. 2007), but distinct molecular features of binding epitopes have generally not resulted from these approaches.
More recent proteome-wide screening techniques have included expressed cDNA libraries (SEREX) (Sahin et al. 1995), protein and peptide arrays (Stockert et al. 1998; Pereira-Faca et al. 2007), random or designed phage displays (Mintz et al. 2003), and more recently self-assembling protein arrays (Ramachandran et al. 2008; Anderson et al. 2008). Cancer-associated auto-antibodies characterized to date have been found to bind intracellular proteins with functions important in cell cycle regulation, such as GPR78 (Mintz et al. 2003), p53 (Lubin et al. 1993), NY-ESO-1, and CDC25 (Liu et al. 2008), but also some cell membrane glycoproteins such as MUC1 (Snijdewint et al. 1999), HER2 (Chapman et al. 2007) and Mesothelin (Hellstrom et al. 2008).
Auto-antibodies are believed to be induced as a result of altered expression of proteins and altered molecular structure due to mutations, alternative splicing and post-translational events such as protein processing and aberrant enzymatic modifications including glycosylation (Anderton 2004; Doyle and Mamula 2005; Doyle and Mamula 2001). These events induce breakage of tolerance and immunity may result. Surprisingly, however, few disease- or more specifically cancer-associated auto-antibody epitopes have been identified and molecularly defined despite considerable efforts and broad proteome screening. This is due to limitations in methods for identification of such auto-antibodies, in that, before the present invention, the appropriate antigen epitopes have not been determined and hence not tested to lead to identification of disease-associated antibodies, to serve as substrates for the detection of disease or to serve as prototype vaccines for induction of immunity against the epitopes of these autoantibodies.
There are few known examples of disease-associated human antibodies to proteins involving glycosylation. One important example is an immunodominant epitope in type II collagen comprising a glycosylated hydroxylysine residue that is involved in collagen-induced arthritis (Backlund et al. 2002). Glycosylation may also modulate protein processing and hence affect exposure of new epitopes as shown in Rasmussen's encephalitis, where an N-glycan blocks proteolysis of a neuronal glutamate receptor and a short preceding peptide epitope (Gahring et al. 2001). Several human monoclonal antibodies have been shown to be directed to epitopes affected by glycosylation (Rauschert et al. 2008; Vollmers and Brandlein 2009; Brandlein et al. 2004a; Brandlein et al. 2004b; Rasmussen and Ditzel 2009), but the nature of the molecular epitopes remains undefined.
There is therefore a need to develop methods for the identification of PTM-containing peptides, such as aberrant glycopeptides (hereinafter “AGP”), that are specifically recognized by disease-associated auto-antibodies. The present invention provides such methods. There is also a need for improved diagnostic tools, such as AGP, that would permit early detection of disease, notably cancer, for example by being used as substrates to capture disease-associated autoantibodies.